2-Step Immunoperoxidase Protocol: Formaldehyde-Fixed Cells and Cytospin Preparations

Preparation of Reagents

Wash Solution

PBS or TBS. TBS wash solution is recommended for phospho antibodies.

Normal Goat Serum Blocking Solution

• 20 ml normal goat serum (Filter sterilize with 0.2 µM filter)
• Volume to 100 ml with PBS or TBS

Store 2-8° C, discard after 3 months.

IHC Antibody Diluent (50mM TBS, 1% BSA)

• 190 ml 1X TBS
• 2 g BSA (Sigma A9647)
• Volume to 200 ml with TBS

Store 2-8° C, discard after 1 year.

May want to use a preservative.

Secondary Antibody

Goat anti-Rabbit IgG Antibody-horseradish peroxidase conjugated (Bethyl cat# A120-201P, A120-401P, A120-501P).

Dilute 1:100 with IHC antibody diluent. Prepare just prior to use. Optimal working dilutions should be determined experimentally by the investigator.

DAB Solution

DAB Reagent set (KPL Cat #54-10-00)

IHC Hematoxylin

• 10 ml Gill 1-regular strength (StatLab #SL97-16))
• 90 ml distilled water

IHC Bluing Solution

• 50ml 10X Tris pH 7-8
• 450 ml distilled water

10% Triton-X 100 Stock

• 10 ml Triton-X 100
• 90 ml dH20

0.25% Triton-X 100

25 µl 10% Triton-X 100

1 ml TBS

Procedure

1. For cells in chambered microscope slides or cells grown on coverslips: Gently rinse off media with TBS - 3 changes for 1 minute each. For cytospins: Allow to air dry after preparation.
2. Formaldehyde fixation - 10-30 minutes
3. Wash Solution - 3 changes for 5 minutes each
4. Circle cytospin with a hydrophobic barrier pen. Do not allow cells to dry for the remaining procedure.
5. Permeabilization step: 0.25% Triton-X 100 - 2-10 minutes. Optimal working dilution and incubation time should be determined experimentally by the investigator.
6. Wash Solution - 3 changes for 5 minutes each
7. Normal goat serum Blocking Solution - 15 minutes
8. Primary Antibody Incubation: 30 minutes - room temperature. Prepare primary antibody with IHC Antibody Diluent. Optimal working dilutions should be determined experimentally by the investigator.
9. Wash Solution - 3 changes for 5 minutes each
10. Secondary Antibody Incubation: 30 minutes - room temperature.
11. Wash Solution - 3 changes for 5 minutes each
12. DAB development - 5-10 minutes. Monitor development with microscope. Do not develop longer than 10 minutes.
13. Wash solution - 3 changes for 5 minutes each
14. IHC Hematoxylin - 1-3 minutes
15. Wash Solution - 3 changes for 5 minutes each
16. IHC Bluing Solution - 1-2 minutes
17. dH20 rinse
18. Remove chamber from microscope slide.
19. 70% reagent alcohol - 3 minutes
20. 95% reagent alcohol; 2 changes for 3 minutes each
21. 100% reagent alcohol; 3 changes for 3 minutes each
22. Xylene - 3 changes for 3 minutes each
23. Mount and coverslip.
24. Place slides flat to dry.